Use of Temperature Sensitive Mutants to Study Yeast DNA Replication

Year: 1984 DOI: 10.7907/gekb-yq20
An improved in vitro DNA replication system in Brij-treated Saccharomyces cerevisiae has been used to screen a random population of temperature-sensitive strains for mutants specifically defective in DNA synthesis. Twenty mutants defective in in vitro DNA synthesis have been isolated. Seven of them fall into three complementation groups -- cdc2, cdc8, and cdc16 -- involved in the control of the cell-division cycle. Because synthesis in vitro represents propagation of replication forks active in in vivo at the time of permeabilization, our findings that cdc2 and cdc16 mutants can incorporate dTMP into DNA in such permeabilized cells at 23°c but not at 37°c supports the conclusion that these two mutations directly affect DNA synthesis. Such an involvement was previously suggested by in vivo analysis for CDC2 but was less clear for CDC16. The usefulness of our screening procedure is further demonstrated by the isolation of replication mutants in previously undescribed complementation groups. One strain shows a serious defect in in vivo DNA synthesis but normal RNA synthesis.

The in vitro system has also been used to purify the CDC8 protein. cdc8 mutant strains are temperature-sensitive for DNA chain elongation and the CDC8 gene product is required for DNA synthesis in vitro in permeabilized yeast cells. Extracts of wild-type A364a yeast restore DNA synthesis in Brij-treated cdc8 mutant. A small, heat-stable protein responsible for this complementation has been partially purified from wild-type cells.

The CDC8 gene has been isolated on recombinant plasmids. The yeast-E. coli shuttle vector YCp50 was used to prepare a recombinant plasmid pool containing the entire yeast genome. Plasmids capable of complementing the temperature-sensitive cdc8-1 mutation were isolated by transformation of a cdc8-1 mutant and selection for clones able to grow at the nonpermissive temperature. The entire complementing activity is carried on a 0.75-kilobase fragment, as revealed by deletion mapping and DNA sequencing. This fragment lies 1 kilobase downstream from the well characterized sup4 gene, a gene known to be genetically linked to CDC8 thus confirming the cloned gene corresponds to the chromosomal CDC8 gene. Two additional recombinant plasmids that complement the cdc8-1 mutation but that do not contain the 0.75-kilobase fragment or any flanking DNA were also identified in this study. These plasmids may contain genes that compensate for the cdc8-1 mutation.

By the following criteria, we have shown that thymidylate kinase, which catalyzes the phosphorylation of thymidine-5'-monophosphate to thymidine-5'-diphosphate in the pathway of synthesis of dTTP from dTMP, is the product of the CDC8 gene. First, transformed strains carrying the CDC8 gene on a stable high-copy-number plasmid express higher levels of both the gene transcript and the kinase activity than does wild type. Secondly, extracts of strains bearing different alleles of cdc8 show no detectable thymidylate kinase activity. Third, the DNA sequence of CDC8 gene reveals an open reading frame that encodes a protein of 216 amino acids with the same amino terminal sequence as thymidylate kinase purified from yeast.